Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: (A) Experimental design, identification of genes and validation plan; (I) Stepwise process leading to identification of genes. We first assessed gene expression in normal and tumor samples in seven different cancers in TCGA dataset and identified deoxyribonucleases that were overexpressed in these cancers. We then evaluated genomic instability in each patient sample by counting the total number of copy number events; data from triple negative breast cancer (TNBC) patients is shown as an example. Genomic instability data was then integrated with expression data to identify deoxyribonucleases that were overexpressed in cancer relative to corresponding normal samples, and whose expression correlated with genomic instability. We thus identified four gene deoxyribonuclease genomic instability signature (E-GIS) representing six human cancers; (II) Bar graph showing number of deoxyribonucleases correlating with genomic instability in each cancer. Four deoxyribonucleases (FEN1, EXO1, EME1 and APE1) correlated with genomic instability in all cancers; (III) Venn diagram showing genomic instability-associated deoxyribonucleases in six human cancers. Separate circles for lung adenocarcinoma (LUAD), prostate adenocarcinoma (PRAD), triple negative breast cancer (TNBC) and one common circle for gastrointestinal (GI) cancers i.e., esophageal adenocarcinoma (EAC), stomach adenocarcinoma (STAD) and pancreatic adenocarcinoma (PAAD) are shown. (IV) Experimental plan for functional validation of identified genes. (B) Knockdown screens to validate E-GIS genes for impact on genome stability and growth of cancer cells. E-GIS genes (APE1, EXO1, FEN1 and EME1) were suppressed in EAC (FLO1) cells using esiRNAs (Millipore/Sigma) and impact on genome stability assessed by evaluating micronuclei (marker of genomic instability) (II) and cell viability (III); CS, control esiRNA; KD, esiRNA-mediated knockdown. (I) Western blot showing knockdown of genes; (II) Images showing nuclei (N) and micronuclei (MN; a marker of genomic instability). X-axis is FITC signal indicating amount of DNA in nuclei or micronuclei and Y-axis (forward scatter) indicates size which distinguishes nuclei from micronuclei; ( III) Bar graph showing relative micronuclei; error bars represent SDs of triplicate experiment. Two-tailed p-values derived by Student t-test (*p < 0.05–0.001; **p < 0.001). (IV) Cell viability assessed using Cell Titer-Glo; CS, control esiRNA; KD, esiRNA-mediated knockdown; error bars represent SDs of triplicate experiment. Two-tailed p-values derived by Student t-test (p ≤0.03).

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Biomarker Discovery, Gene Expression, Expressing, Functional Assay, Knockdown, Marker, Control, esiRNA, Western Blot, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: (A-B) APE1 is overexpressed in solid tumors. Relative expression (Log2) of APE1 in TCGA datasets was plotted using R and normal and tumor samples compared with limma software. EAC, esophageal adenocarcinoma; HPBC, hormone positive breast cancer; TNBC, triple negative breast cancer; LUAD; lung adenocarcinoma; PRAD, prostate adenocarcinoma; COAD, colon adenocarcinoma. (B) APE1 is overexpressed in EAC patient samples; Panels: (I) APEX1 expression in frozen tissue specimens of normal squamous epithelium (NSE), Barrett’s esophagus (BE), dysplasia and EAC detected by immunohistochemistry; (II) APEX1 expression in EAC and normal esophageal tissue specimens on a tissue array (Biomax); (C-E) Impact on genomic instability: (C) APE1 was suppressed in cancer cell lines (FLO-1 and OE19, esophageal adenocarcinoma; MCF7, breast cancer; A549, epithelial lung carcinoma; PC3, prostate adenocarcinoma) using lentiviral shRNAs and right after selection, live cell fractions evaluated for micronuclei (marker of genomic instability) using flow cytometry. Panels: (I) Representative images showing nuclei (N) and micronuclei (MN). X-axis is FITC signal indicating amount of DNA in nuclei or micronuclei and Y-axis (FSC or forward scatter) indicates size which distinguishes nuclei from micronuclei; (II) Bar graph showing relative micronuclei in control and knockdown cells; Error bars represent SDs of four independent experiments. Two-tailed p-values for significance of difference between control and each treated sample was derived by Welch’s t-test (*p < 0.05–0.001; **p < 0.001). CS, control shRNA; KD, cells transduced with APE1 shRNA; (D) All five cell lines (described above) were treated with API3 (1.5 ⍰M) for 48 h and live cell fractions evaluated for micronuclei (marker of genomic instability) using flow cytometry. Panels: (I) Representative images showing nuclei (N) and micronuclei (MN); (II) Bar graph showing relative micronuclei in control and treated cells; C, control (DMSO) treated cells; API3, API3-treated cells; For FLO-1, OE19 and MCF7 error bars represent SDs of four independent experiments and two-tailed p-values for significance of difference between control and each treated sample derived by Welch’s t-test (p < 0.03). Bar graphs for A549 and PC3 cell lines represent two independent experiments; (E) Cancer cell lines, esophageal adenocarcinoma (OE19. top panel) and epithelial lung carcinoma (A549; bottom panel) were treated with APE1 inhibitor (API3; 1.5 ⍰M), cisplatin (CIS; 5 ⍰M) or combination of both and live cell fractions evaluated for micronuclei (marker of genomic instability) using flow cytometry. Panels: (I) Representative images showing nuclei (N) and micronuclei (MN); (II) Bar graph showing fold change in micronuclei, relative to control cells.

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Expressing, Software, Immunohistochemistry, Selection, Marker, Flow Cytometry, Control, Knockdown, Two Tailed Test, Derivative Assay, shRNA, Transduction

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: (A) Chemical or transgenic suppression of APE1 reduces cisplatin-induced DNA breaks in solid cancer cells. (I) Cancer cell lines (FLO-1, esophageal adenocarcinoma; A549, epithelial lung carcinoma; MCF7, breast cancer) were treated with APE1 inhibitor (API3; 1.5 ⍰M), cisplatin (5 ⍰M) or combination of both and live cell fractions evaluated for expression of APE1 and γ -H2AX (marker of DNA breaks) by Western blotting. Lanes: 1, control; 2, API3; 3, cisplatin; 4, API3+cisplatin. (II) Control and APE1-knockdown cancer cells (OE19, esophageal adenocarcinoma; A549, epithelial lung carcinoma) were treated with cisplatin (15 ⍰M) and expression of APE1 and γ -H2AX assessed by Western blotting. Lanes: 1, control shRNA; 2, control shRNA treated with cisplatin; 3, APE1-shRNA; 4, APE1-shRNA treated with cisplatin; (B-C) Chemical or transgenic inhibition of APE1 inhibits RAD51 expression and HR activity. (B) EAC ( FLO-1) cells treated with API3 were evaluated for RAD51 expression by Western blotting (I), RAD51 promoter activity using a plasmid in which RAD51 drives luciferase expression (II), and HR activity using the plasmidbased assay (III). (C) EAC (FLO-1) cells were treated with shRNAs (CS, control shRNAs; KD, cells treated with APE1-targeting shRNAs). Panels: (I) APE1 expression evaluated by Western blotting; (II) HR activity assessed by plasmid-based assay; (III) Expression of RAD51 (top panel) and its phosphorylated form (bottom panel) evaluated by Western blotting; (D) Dose-dependent inhibition of HR activity by APE1 inhibitor in solid tumor cell lines. Cancer cell lines (OE19, esophageal adenocarcinoma; MCF7, breast cancer; A549, epithelial lung carcinoma; PC3, prostate adenocarcinoma) were treated with APE1 inhibitor (API3) at different concentrations for 48 HR and impact of HR activity determined using plasmid-based functional assay described in Methods.

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Transgenic Assay, Expressing, Marker, Western Blot, Control, Knockdown, shRNA, Inhibition, Activity Assay, Plasmid Preparation, Luciferase, Functional Assay

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: (A) APE1-knockdown inhibits growth of solid cancer cell lines. APE1 was suppressed in human cancer cell lines - esophageal adenocarcinoma (FLO-1, OE19), breast cancer (MCF7), epithelial lung carcinoma (A549) and prostate adenocarcinoma (PC3) using shRNAs and impact on cell viability assessed using Cell Titer-Glo; Control, control shRNA; APE1-KD (knockdown), APE1 shRNA; error bars indicate SDs of triplicate experiment. Two-tailed p values for significance of difference between control and knockdown cells at day 7 ranged from 0.015 to 0.0007. (B) APE1 inhibitor increases cytotoxicity of cisplatin in vitro . Cancer cell lines (MCF7, A549 and FLO-1) were treated with APE1 inhibitor (API3), alone as well as in the presence of chemotherapeutic agent cisplatin, and cell viability measured after 48 hr. Bar graphs of cells treated with API3 with cisplatin are shown; error bars represent SDs of three experiments. Combination index plots (calculated using CalcuSyn software) are shown in Supplementary Figure 8. (C) APE1 inhibitor inhibits cancer cell growth and increases cytotoxicity of cisplatin in vivo . EAC (OE19) cells were injected subcutaneously in SCID mice and following appearance of tumors, mice treated with either DMSO, API3 (12 mg/kg, daily for 2 weeks), cisplatin (3 mg/kg, once a week for 2 weeks) or combination of both drugs. Line plots showing tumor growth in control and treated mice (n = 5 each); error bars represent SDs.

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Knockdown, Control, shRNA, Two Tailed Test, In Vitro, Software, In Vivo, Injection

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: Normal primary human esophageal epithelial cells (HEsEpi; ScienCell) were transfected with plasmids carrying empty control vector (C) or APE1 under a strong promoter (APE1O) and evaluated for various parameters of genome stability. (A) DNA breaks and homologous recombination (HR). (I) Western blot showing expression of APE1, RAD51 and γ-H2AX (marker of DNA breaks) in control and APE1-overexpressing cells, evaluated right after selection; (II) DNA breaks assessed by Comet assay right after selection; (III) HR activity evaluated by plasmid-based assay; error bars represent SD of three experiments and two tailed p value is < 0.0002; (B) Impact on centrosome: Centrosome investigated three weeks after selection. Arrows show multiple centrosomes clustered together in APE1O cells; (C) Impact on karyotype: Karyotypes of control ( I ) and APE1O ( II-III ) cells examined after 60 days in culture. Panels II and III are two different examples of karyotypes. Mitotic index (MI) of APE1O = 3.9%; 9-12 chromosome aberrations (arrowheads on representative translocations); (D) Impact on mutational frequency evaluated by WGS: Normal primary esophageal epithelial cells carrying control plasmid (C) and that for APE1-overexpression (APE1O) were cultured for sixty days and analyzed for new mutations, relative to baseline (day 0) cells, using WGS. (I) New mutations in control and APE1O cells identified by 3 different software tools; (II) Substitution types in APE1-overexpressing cells throughout genome are presented in context of the sequence immediately 5’ and 3’ to the mutated base; (III) Fraction of contribution of each mutation type at each sequence context for the mutational signatures. For investigation of mutational signatures or processes, SNPs were removed to ensure that somatic mutation callers do not confuse about the SNPs and SNVs. After removing SNPs, there were 44 mutations in control vs. 3500 mutations in APE1-overexpression cells.

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Transfection, Control, Plasmid Preparation, Homologous Recombination, Western Blot, Expressing, Marker, Selection, Single Cell Gel Electrophoresis, Activity Assay, Two Tailed Test, Over Expression, Cell Culture, Software, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

doi: 10.1101/2022.04.20.488830

Figure Lengend Snippet: (A) APE1 regulates G2/M checkpoint in EAC cells. (I) Expression profile showing common pathways which are upregulated following APE1-overexpression in normal esophageal epithelial (HEsEpi) cells, whereas downregulated in EAC (FLO1) cells treated with APE1 inhibitor (API3); cell cycle (G2/M) is the most significant pathway impacted. (II-IV) FLO-1 cells, untreated and treated with API3 (2 ⍰M), were evaluated for cell cycle; cell cycle profiles (II-III) and bar graph showing percentage of cells in different phases of cell cycle (IV) are presented; error bars indicate SDs of triplicate experiment. Two-tailed p value for significance of difference in G2 phase between control and treated cells derived by Student’s t test is 1.5×10 -5 . (B) Impact of APE1 inhibitor on G2/M checkpoint in different cancers. Human cancer cell lines - esophageal adenocarcinoma (OE19), breast cancer (MCF7), epithelial lung carcinoma (A549) and prostate adenocarcinoma (PC3), untreated (control) and treated with API3 (1.5 ⍰M), were evaluated for cell cycle; Panels: (I-IV) Cell cycle profiles; (V) Bar graph showing percent increase in G2 fraction relative to control cells for all cancer cell lines.

Article Snippet: Affinity purified human APE1 antibody (NB100-101), Novus Biologicals LLC, Centennial, CO; FEN1 Antibody (2746s), Cell Signaling Technology, Inc., Danvers, MA; EXO1 antibody (A302-639A), Bethyl Laboratories, Montgomery, TX, USA; EME1 antibody (PA5-101988), Invitrogen, Carlsbad, CA, USA; RAD51 antibody (ab176458), Abcam, Cambridge, MA, USA; RPA32(A300-244A), Bethyl Laboratories, Montgomery, TX, USA; RPA32 (phospho Ser4 and phospho Ser8) antibody, Novus Biologicals LLC, Centennial, CO; Anti-phospho-H2A.X (Ser139) antibody, (07-164), Sigma-Aldrich, St. Louis, MO, USA; GAPDH (14C10) rabbit monoclonal antibody ( #2118), Cell Signaling Technology, Inc., Danvers, MA; ß-Tubulin antibody (#2146), Cell Signaling Technology, Inc., Danvers, MA.

Techniques: Expressing, Over Expression, Two Tailed Test, Control, Derivative Assay

Journal: Biomedicines

Article Title: Plasma APE1/Ref-1 Correlates with Atherosclerotic Inflammation in ApoE −/− Mice

doi: 10.3390/biomedicines8090366

Figure Lengend Snippet: Correlation analysis of hematologic parameters with plasma APE1/Ref-1 levels.

Article Snippet: To establish a standard curve, purified recombinant human APE1/Ref-1 (MediRedox, Daejeon, Korea) was serially diluted (2-fold) and used in a concentration series from 0.312–20 ng/mL.

Techniques: Clinical Proteomics, Western Blot